Acetyl Coenzyme A carboxylase (ACCase) is the rate-limiting enzyme in the fatty acid biosynthesis pathway in plant, animal, yeast, and bacterial cells. Structurally, ACCases are biotinylated and are large enzymes consisting of two or more subunits. For example, most ACCases of animals, the cytoplasmic version in plants, and yeast are dimers of 420 to 700 kD native MW and contain subunits of 200 to 280 kD. Higher plant and algal plastid, and bacterial ACCases are 700 to 740 kD complexes 20 to 180 kD subunits.
Acetyl CoA Carboxylase (ACCase) catalyzes the formation of malonyl-CoA from acetyl-CoA and bicarbonate in animal, plant, and bacterial cells. Malonyl-CoA is an essential substrate for (i) de novo fatty acid (FA) synthesis, (ii) fatty acid elongation, (iii) synthesis of secondary metabolites such as flavonoids and anthocyanins, and (iv) malonylation of some amino acids and secondary metabolites. Synthesis of malonyl-CoA is the first committed step of flavonoid and fatty acid synthesis and current evidence suggests that ACCase catalyzes the rate-limiting step of fatty acid synthesis. Formation of malonyl-CoA by ACCase occurs via two partial reactions and requires a biotin prosthetic group:
(i) Enzyme-biotin+ATP+HCO3→Enzyme-biotin-CO, +ADP+Pi
(ii) Enzyme-biotin-CO2+Acetyl-CoA→Enzyme-biotin+malonyl CoA
The net reaction is:Acetyl CoA+ATP+HCO3→malonyl-CoA+ADP+Pi
In E. coli, these reactions are catalyzed by three distinct components; biotin carboxylase, biotin-acetyl CoA transcarboxylase, and biotin carboxyl carrier protein, which can be separated and yet retain partial activity. Plant and animal cytoplasmic ACCases contain all three activities on a single polypeptide.
Two different forms of the ACCase complex exist in plants (as described, for example, in Sasaki, Y. and Nagano, Y. (2004) Biosci. Biotechnol. Biochem. 68(6):1175-1184); the cytoplasmic enzyme, consisting of a very large single polypeptide chain, and the plastidic ACCase complex. The plastidic complex is a multi-enzyme complex composed of biotin carboxyl carrier protein (BCCP), biotin carboxylase, and a carboxyltransferase complex made up of two pairs of α and β subunits.
Several pieces of evidence indicate that, at least in higher plants, the chloroplast ACCase complex is subject to control via post-translational modification. Kozaki and Sasaki Biochem J., 339:541 (1999) describe light levels and the addition of reducing agent (dithiothreitol) as being able to increase chloroplast ACCase activity, while the amount of ACCase protein remained roughly unchanged.
Savage and Ohlrogge, Plant J., 18:521 (1999) described purification of pea chloroplast ACCase complex, and showed that the β-subunit of the complex was phosphorylated in vivo. Removal of the phosphates by phosphatase treatment dramatically reduced the ACCase activity in the sample.
Under certain physiological conditions, mammalian ACC activity is rapidly regulated by reversible phosphorylation (for example, as described in Kim, K.-H. (1983) Curr. Top. Cell Regul., 22, 143-176; and Kim, K.-H., et al., FASEB J. (1989) 3, 2250-2256) which involves specific protein kinases that phosphorylate and inactivate ACC (for example, as described in Kim, K.-H., et al., FASEB J. (1989) 3, 2250-2256), and phosphatases that dephosphorylate and activate the enzyme.
Ha, J. et al. (The J. of Biol. Chem. (1994) 269 (35) pp. 22162-22168) created and expressed a cDNA of the entire coding region of the rat Acetyl-CoA carboxylase and identified eight different phosphorlyation sites on the carboxylase molecule. The sites were identified by comparing phosphopeptide sequences and the deduced amino acid sequences from rat ACC cDNA (for example, as described in Lopez-Casillas, F., et al. (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 5784-5788; Munday, M. R., et al. (1988) Eur. J. Biochem., 175, 331-338; Haystead, T. A. J. and Hardie, D. G. (1988) Eur. J. Biochem., 175, 339-345; and Haystead, T. A. J. et al. (1988) Eur. J. Biochem., 175, 347-354). The identified sites are Ser 23, 25, 29, 77, 79, 95, 1200, and 1215. The roles of these phosphorylation sites on the activation of ACCase are not well understood.
Increasing the amount of ACCase activity in the cell has been proposed as a mechanism to increase the lipid content (for example, TAG, DAG, and other acyl lipids) in algae, higher plants, yeast, and mammals. Attempts have been made to increase ACCase activity by increasing the amount of protein present via upregulation of a native ACCase gene or by introduction of a transgene under a stronger promoter. These efforts have produced increased levels of ACCase protein in the target organisms, but have not significantly altered lipid level (for example, as described in Hu et al., The Plant J., 54:621 (2008)).
In order to increase fatty acid synthesis in a cell, what is needed is not simply to increase production of an ACCase protein, but rather to increase the level of ACCase activity in the cell, resulting in an increase in lipid production. The present disclosure meets that need.